Phospholemman (PLM), the 1st member of the FXYD family of small integral membrane proteins involved in regulation of ion transport, modulates the function of both Na+-K+-ATPase and Na+/Ca2+ exchanger (NCX1) in the heart. In the current grant period, using 3 model systems (adenovirus-mediated gene transfer in adult rat myocytes, transfected HEK293 cells, and PLM knockout (KO) mice), and different techniques (electrophysiology, biochemistry, radioactive tracer uptake, and site-directed mutagenesis), we have unequivocally established that PLM directly regulates NCX1 function, independent of its effects on Na+-K+- ATPase. We showed physical association between the cytoplasmic tails of PLM with the proximal intracellular loop of NCX1. Under conditions in which [Ca2+]o is raised to favor Ca2+ influx via NCX1, PLM regulates cardiac contractility mainly by modulating NCX1 rather than Na+-K+-ATPase activity. By contrast, when myocytes are Na+ loaded by rapid pacing and isoproterenol treatment, PLM limits inotropic response to b-adrenergic stimulation by enhancing Na+-K+-ATPase activity. Under baseline conditions, the effects of PLM on either Na+- K+-ATPase or NCX1 are not apparent. These results lead us to 2 hypotheses: (1) there is a small stretch of amino acid residues in the proximal linker domain of NCX1 that interacts with phosphorylated PLM; and (2) in the intact heart subjected to stress and therefore high catecholamine levels, PLM is phosphorylated at serine68 which simultaneously accelerates Na+-K+-ATPase but retard Na+/Ca2+ exchange activities. This coordinated action of PLM is necessary to minimize arrhythmogenesis and preserve inotropic response under b-adrenergic stimulation. In this competitive grant renewal, we wish to test our hypotheses by: (1) mapping out the exact sites/residues in NCX1 that are critically involved in its interaction with PLM; and (2) evaluate the mechanism by which PLM regulates cardiac contractility in hearts in vivo, both under resting and stressful conditions. We will use a combination of in vivo (echocardiography, catheterization, transduction of exogenous genes by rAAV9 injection) and in vitro (patch-clamp, single myocyte contractility and Ca2+ and Na+ measurements, co-immunoprecipitation and GST pulldown) techniques, focusing on 2 experimental model systems: transfected HEK293 cells and PLM-KO mice with inducible transgene (TG). We already have 2 genetically engineered mice (PLM-KO, inducible PLMS68E TG) on hand, and we are confident that given the resources, we will be able to generate inducible PLMS68E TG in PLM-KO background for our studies. We have found a NCX1 mutant (248-252 AAAAA) that exhibits normal NCX1 current but is not inhibited by PLM. By expressing this NCX1 mutant in NCX1-KO hearts (Dr. Kenneth Philipson has agreed to provide us the cardiac-specific NCX1-KO mouse), we should be able to critically test the physiological relevance of NCX1 regulation by PLM. We are the only laboratory that focuses on PLM and NCX1 interactions in the heart. We have all the reagents and techniques at hand and we believe we are well situated to critically examine the mechanism by which PLM regulates cardiac contractility. Alterations in phospholemman expression or its phosphorylation state are observed in ischemic cardiomyopathy and schizophrenia. In addition, the 2 classes of drugs (b-blockers and ACE inhibitors) that have been clinically found to be efficacious in congestive heart failure and ischemic heart disease may have PLM as a common target. Understanding how PLM regulates cardiac contractility in health and disease is therefore paramount for newer and rational therapy to be designed. PUBLIC HEALTH RELEVANCE: Phospholemman regulates 2 important ion transporters in the heart. This proposal uses 2 different genetically altered mice specifically engineered to critically evaluate whether regulation of sodium-calcium exchanger or sodium-potassium pump is the major mechanism by which phospholemman affects heart contraction. In addition, the exact molecular site in sodium-calcium exchanger interacting with phospholemman will be mapped out by mutagenesis.